Bap-1 proteins

ABSTRACT

The present invention provides the amino acid and nucleotide sequence of a protein that binds to  beta 3 integrins,  alpha IIb and Src kinase and is involved in integrin mediated signaling. Based on this disclosure, the present invention provides methods for identifying agents that block integrin mediated signaling, methods of using agents that block integrin mediated signaling to modulate biological and pathological processes, and agents that block integrin mediated signaling.

RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/753,038, filed Nov. 18, 1996, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of integrin-mediatedsignaling, particularly signal transduction mediated by β3 integrinssuch as αVβ3 and αIIbβ3. The invention relates specifically to theidentification of a novel human gene, tentatively named Bap-1. Bap-1encodes a protein, Bap-1, that interacts with the cytoplasmic domains ofαIIb or β3 integrins, and Src kinase, and is involved in β3integrin-mediated signal transduction.

BACKGROUND OF THE INVENTION

Integrins are a family of αβ heterodimers that mediate adhesion of cellsto extracellular matrix proteins and to other cells (Clark et al.,Science 268: 233-239, 1995). Integrins also participate in signaltransduction, as evidenced by either an alteration in adhesive affinityof cell surface integrins in response to cellular activation (termedinside-out signal transduction) or by affecting intracellular signalingpathways following integrin-mediated adhesion (termed outside-in signaltransduction). Many biological responses are dependent at least to someextent upon integrin-mediated adhesion and cell migration, includingembryonic development, hemostasis, clot retraction, mitosis,angiogenesis, cell migration, inflammation, immune response, leukocytehoming and activation, phagocytosis, bone resorption, tumor growth andmetastasis, atherosclerosis, restenosis, wound healing, viralinfectivity, amyloid toxicity, programmed cell death and the response ofcells to mechanical stress.

The integrin family consists of 15 related known α subunits (α1, α2, α3,α4, α5, α6, α7, α8, α9, αE, αV, αIIb, αL, αM, and αX) and 8 relatedknown β subunits (β1, β2, β3, β4, β5, β6 β7, and β8). (Luscinskas etal., FASEB J. 8:929-938, 1994.) Integrin α and β subunits are known toexist in a variety of pairings. Integrin ligand specificity isdetermined by the specific pairing of the α and β subunits, althoughsome redundancy exists as several of the integrins are known to bind thesame ligand. Most integrins containing the β1, β2, β3, β5, β6, and β7subunits have been found to transduce signals (reviewed by Hynes, Cell69:11-25, 1992). Integrins are involved in both "inside-out" and"outside-in" signaling events.

Various pathologies associated with integrin-related defects are known.For example, inherited deficiencies of GP IIb-IIIa (also termed αIIbβ3)content or function have been described (termed Glanzmann'sthrombasthenia) and are characterized by platelets that do not bindadhesive proteins and therefore fail to aggregate, resulting in alife-long bleeding diathesis. Inhibitors of the binding of fibrinogenand von Willebrand factor to GP IIb-IIIa have been described and havebeen found to block platelet aggregation in vitro and to inhibitclinical thrombosis in vivo (The EPIC Investigators, New England Journalof Med. 330:956-961, 1994; J. E. Tcheng et al., Circulation91:2151-2157, 1995). Also, leukocyte adhesion deficiency (LAD) resultsfrom the absence of a β2 subunit, and is characterized by leukocyteswhich fail to bind β2 integrin ligands, resulting in individuals thatare susceptible to infections.

The most studied platelet integrin αIIbβ3 (GPIIbIIIa) plays a criticalrole in homeostasis (platelet aggregation) and also in thrombosis. TheαVβ3 plays a critical role in melanoma metastasis and angiogenesis,which is essential for cancer cell growth. The adhesion capacity ofαIIbβ3 is known to be stimulated by various agonists such as thrombin,collagen, and ADP. This is termed inside-out signaling. There isaccumulating evidence suggesting that integrins, in various cells andtissues including platelets, are also capable of mediating signals fromthe exterior to the cell interior, and that these signals can triggercellular processes such as stimulating protein tyrosine phosphorylation,activating Na+/H+ antiporter, assembly of cytoskeletal structures andregulating gene expression that is involved in cell migration andproliferation. However, the mechanisms by which these signals aretransmitted remain elusive. It has been hypothesized that thecytoplasmic tails of αIIbβ3 and other integrins may play important rolesin adhesion by modulating the ligand-binding function of theextracellular domains through responses to intracellular signalsgenerated by agonists stimulation (inside-out), and by mediating signalstriggered by integrin receptor occupancy to intracellular molecules thatmay play a pivotal role in cellular physiological and pathologicalfunctions (outside-in).

A. Inside-Out Signaling

Inside-out signal transduction has been observed for β1, β2, and β3integrins. (R. O. Hynes, Cell 69:11-25, 1992; D. R. Phillips, et al.Cell 65:359-362, 1991, S. S. Smyth et al., Blood 81:2827-2843, 1993; M.H. Ginsberg, et al. Thromb. Haemostasis 70:87-93, 1993; R. L. Julianoand S. Haskill, J. Cell Biol. 120:577-585, 1993; E. Rouslahti, J. Clin.Invest. 87:1-5, 1991; Weber et al., J. Cell Biol. 134:1063-1073, 1996.)

Perhaps the most widely studied integrin that is involved in inside-outsignaling is GP IIb-IIIa, the receptor for four adhesive proteins,fibrinogen, von Willebrand factor, vitronectin and fibronectin that bindto stimulated platelets (D. R. Phillips, et al., Blood 71:831-43, 1988).The binding of adhesive proteins to GP IIb-IIIa is required for plateletaggregation and normal hemostasis and is also responsible for occlusivethrombosis in high shear arteries.

GP IIb-IIIa is known to be involved in inside-out signal transductionbecause GP IIb-IIIa on the surface of unstimulated platelets is capableof recognizing only immobilized fibrinogen. In response to plateletstimulation by agents such as thrombin, collagen and ADP, GP IIb-IIIabecomes a receptor for the four adhesive proteins identified in theprevious paragraph, and the binding of fibrinogen and von Willebrandfactor causes platelets to aggregate. A monoclonal antibody has beendescribed which detects the activated, receptor competent state of GPIIb-IIIa, suggesting that the conformation of the receptor competentform of GP IIb-IIIa differs from that of GP IIb-IIIa which does not bindsoluble fibrinogen or von Willebrand factor (S. J. Shattil, et al., J.Biol. Chem. 260:11107-11114, 1985). It has been postulated thatinside-out GP IIb-IIIa signal transduction is dependent on cellularproteins that act to repress or stimulate GP IIb-IIIa activation (M. H.Ginsberg, et al., Curr. Opin. Cell Biol. 4:766-771, 1992).

β2 integrins on leukocytes also respond to inside-out signaltransduction which accounts, for example, for the increased bindingactivity of LFA-1 (αLβ₂) on stimulated lymphocytes and the increasedbinding activity of MAC-1 (αmβ₂) on stimulated neutrophils (reviewed byT. Springer, Curr. Biol. 4:506-517, 1994).

B. Outside-In Signaling

Most integrins can be involved in outside-in signal transduction asevidenced by observations showing that binding of adhesive proteins orantibodies to integrins affects the activities of many cells, forexample cellular differentiation, various markers of cell activation,gene expression, and cell proliferation (R. O. Hynes, Cell 69:11-25,1992). The involvement of GP IIb-IIIa in outside-in signaling isapparent because the binding of unstimulated platelets to immobilizedfibrinogen, a process mediated by GP IIb-IIIa, leads to plateletactivation and platelet spreading (N. Kieffer and D. R. Phillips, J.Cell Biol. 113:451-461, 1991; Haimovich et al., J. Biol. Chem.268:15868-15877, 1993).

Outside-in signaling through GP IIb-IIIa also occurs during plateletaggregation. Signaling occurs because fibrinogen or von Willebrandfactor bound to the activated form of GP IIb-IIIa on the surface ofstimulated platelets, coupled with the formation of platelet-plateletcontacts, causes further platelet stimulation through GP IIb-IIIa signaltransduction. In this manner, binding of adhesive proteins to GPIIb-IIIa can both initiate platelet stimulation or can augmentstimulation induced by the other platelet agonists such as ADP, thrombinand collagen. The binding of soluble fibrinogen to GP IIb-IIIa onunstimulated platelets can also be induced by selected GP IIb-IIIaantibodies such as LIBS6 (M-M. Huang et al., J. Cell Biol. 122:473-483,1993): although platelets with fibrinogen bound in this manner are notbelieved to be stimulated, such platelets will aggregate if agitated andwill become stimulated following aggregation through GP IIb-IIIa signaltransduction.

Outside-in integrin signal transduction results in the activation of oneor more cascades within cells. For GP IIb-IIIa, effects caused byintegrin ligation include enhanced actin polymerization, increased Na⁺/H⁺ exchange, activation of phospholipases, increased phosphatidylturnover, increased cytoplasmic Ca⁺⁺, and activation of kinases. Kinasesknown to be activated include PKC, myosin light chain kinase, src, sykand pp125FAK. Kinase substrates identified include pleckstrin, myosinlight chain, src, syk, pp125FAK, and numerous proteins yet to beidentified (reviewed in E. A. Clark and J. S. Brugge, Sci. 268:233-239,1995). Many of these signaling events, including phosphorylations, alsooccur in response to ligation of other integrins (reviewed in R. O.Hynes, Cell 69:11-25, 1992). Although these other integrins havedistinct sequences and distinct α-β parings that allow for ligandspecificity, the highly conserved nature of the relatively smallcytoplasmic domains, both between species and between subunits, predictsthat related mechanisms will be responsible for the transductionmechanisms of many integrins.

C. Signal Transduction

The involvement of the cytoplasmic domain of GP IIb-IIIa in integrinsignal transduction is inferred from mutagenesis experiments. Deletionof the cytoplasmic domain of GP IIb results in a constitutively activereceptor that binds fibrinogen with an affinity equivalent to thewild-type complex, implying that the cytoplasmic tail of GP IIb has aregulatory role (T. E. O'Toole, et aL, Cell Regul. 1:883-893, 1990).Point mutations, deletions and other truncations of GP IIb-IIIa affectsthe ligand binding activity of GP IIb-IIIa and its signaling response(P. E. Hughes, et al., J. Biol. Chem. 270:12411-12417, 1995; J. Ylanne,et al., J. Biol. Chem. 270:9550-9557, 1995).

Chimeric, transmembrane proteins containing the cytoplasmic domain of GPIIIa, but not of GP IIb, inhibit the function of GP IIb-IIIa (Y. P. Chenet al., J. Cell Biol. 269:18307-18310, 1994), implying that free GP IIIacytoplasmic domains bind proteins within cells which are necessary fornormal GP IIb-IIa function. Several proteins have been shown to bindeither the transmembrane domains or the cytoplasmic domains of GP IIb orGP IIIa.

CD-9, a member of the tetraspanin family of proteins (F. Lanza, et al.,J. Biol. Chem. 266:10638-10645, 1991), has been found to interact withGP IIb-IIIa on aggregated platelets. β3-endonexin, a protein identifiedthrough two hybrid screening using the cytoplasmic domain of GP IIIa asthe "bait", has been found to interact directly and selectively with thecytoplasmic tail of GP IIIa (S. Shattil et al., J. Cell. Biol.131:807-816, 1995). β3-endonexin shows decreased binding to the GP IIIacytoplasmic domain containing the thrombasthenic S752-P mutation. It isnot yet known whether either of these GP IIIa-binding proteins areinvolved in signal transduction.

Cytoplasmic proteins that bind to αVβ3 have also been described whichmay be interacting with the integrin at the GP IIIa cytoplasmic domainsequence. Bartfeld and coworkers (N. S. Bartfeld et al., J. Biol. Chem.268:17270-17276, 1993) used immunoprecipitation from detergent lysatesto show that a MW=190-kDa protein associates with the αVβ3 integrin fromPDGF-stimulated 3T3 cells. IRS-1 was found to bind to the αVβ3 integrinfollowing insulin stimulation of Rat-1 cells stably transfected with DNAencoding the human insulin receptor (K. Vuori and E. Ruoslahti, Sci.266:1576-1578, 1994). Kolanus et al. (Cell 86:233-242, 1996) recentlyidentified Cytohesin-1. Cytohesin-1 specifically binds to theintracellular portion of the integrin β2 chain, and overexpression ofcytohesin-1 induces β2 integrin-dependent binding of Jurkat cells toICAM-1. A novel serine/threonine kinase, ILK-1, was found to associatewith the β1 cytoplasmic domain (Hannigan et al., Nature 379:91-96,1996). Overexpression of ILK-1 inhibits adhesion to the integrin ligandsfibronectin, laminin, and vitronectin.

Integrin binding to adhesive proteins and integrin signal transductionhave a wide variety of physiological roles, as identified above.Enhanced signaling through integrins allows for increased cell adhesionand activation of intracellular signaling molecules which causesenhanced cell mobility and growth, enhanced cell responsiveness, andmodulations in morphological transformations. Although integrinsresponsible for cellular function have been described and signalingevents are beginning to be elucidated, the mechanism by which integrinstransduce signals remains to be determined.

To understand the molecular mechanisms of the inside-out and outside-insignaling roles mediated by the cytoplasmic tails of β3 integrinrequires the identification of the intracellular molecules that interactwith the intracellular tails of integrin. It has been reported thatα-actinin binds to β1 tails in vitro (Otey et al. J. Biol. Chem.268:21193-21197, 1993) but the functional relevance of these bindings isnot clear. By using yeast two-hybrid, ILK-1 was identified as a β1interacting protein but ILK-1 does not bind to β3 (Hannigan et al.,Nature 379:91-96 (1996). The present invention describes the molecularcloning of a novel human gene, Bap-1, encoding a protein, Bap-1, thatassociates with the integrin subunit αII and β3 cytoplasmic tails. Bap-1was also found to associate with Src kinase. The molecular isolation ofBap-1 forms the basis for the development of therapeutic agents thatmodulate integrin-mediated signal transduction.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the isolation andidentification of a protein that binds to the cytoplasmic domain of theβ3 subunit of integrins, hereinafter Bap-1 or the Bap-1 protein. Bap-1was subsequently demonstrated to associate with the cytoplasmic domainof the αIIb subunit and Src kinase. Based on this observation, thepresent invention provides purified Bap-1 protein, useful in a varietyof ways because of such associations.

The present invention further provides nucleic acid molecules thatencode the Bap-1 protein. Such nucleic acid molecules can be in anisolated form, or can be operably linked to expression control elementsor vector sequences.

The present invention further provides methods of identifying othermembers of the Bap-1 and/or Bap family of proteins. Specifically, thenucleic acid sequence of Bap-1 can be used as a probe, or to generatePCR primers, in methods to identify nucleic acid molecules that encodeother members of the Bap-1 or Bap family of proteins.

The present invention further provides antibodies that bind to Bap-1.Such antibodies can be either polyclonal or monoclonal. Anti-Bap-1antibodies can be used in a variety of diagnostic formats and for avariety of therapeutic methods.

The present invention further provides methods for reducing or blockingthe association of an integrin with a cytoplasmic signaling partner.Specifically, the association of an integrin with a cytoplasmicsignaling partner, such as Bap-1 or a Bap-1/signaling partner complex,e.g., Bap-1/Src kinase, can be blocked or reduced by contacting anintegrin having a β3 or αIIb subunit with an agent that blocks thebinding of Bap-1 or the Bap-1/signaling partner complex to the integrin.The method can utilize an agent that binds to the cytoplasmic domain ofthe integrin or an agent that binds to Bap-1 or the Bap-1/signalingpartner complex such as the Bap-1/Src complex.

Blocking integrin/Bap-1 associations can be used to modulate biologicaland pathological processes that require integrin mediated signals. Suchmethods and agents can be used to modulate cellular attachment oradhesion to a substrate or another cell, cellular migration, cellularproliferation and cellular differentiation. Pathological processesinvolving these actions include thrombosis, inflammation, tumormetastasis, wound healing and others noted above.

The present invention further provides methods for isolating integrinsignaling partners that bind to Bap-1 or to a Bap-1 /β3 complex.Integrin signaling partners are isolated using the Bap-1 protein or theBap-1 /β3 complex as a capture probe. Specifically, the Bap-1 protein,or a fragment thereof, or the Bap-1 /β3 complex, is mixed with anextract prepared from an integrin expressing cell under conditions thatallow association of the Bap-1 protein, fragment, or complex with asignaling partner. Non-associated cellular constituents are removed fromthe mixture and the signaling partner is released from the captureprobe. Alternatively, Bap-1 can be used as bait in the yeast two-hybridsystem to screen an expression library and identify genes that encodeproteins with the ability to bind to Bap-1 protein. Signaling partnersisolated by these methods are useful in preparing antibodies and alsoserve as targets for drug development.

The present invention further provides methods to identify agents thatcan block or modulate the association of an integrin with Bap-1 or asignaling complex. Specifically, an agent can be tested for the abilityto block, reduce or otherwise modulate the association of an integrinwith Bap-1 or a signaling complex by incubating the Bap-1 protein, or afragment thereof, with a β3 integrin and a test agent and determiningwhether the test agent blocks or reduces the binding of the Bap-1protein to the β3 integrin. Agonists, antagonists and other modulatorsexpressly are contemplated.

The biological and pathological processes that require Bap-1/integrininteraction can further be modulated using gene therapy methods.Additional genetic manipulation within an organism can be used to alterthe expression of a Bap-1 gene or the production of a Bap-1 protein inan animal model. For example, a Bap-1 gene can be introduced into anindividual deficient for Bap-1 to correct a genetic deficiency; peptidemodulators of Bap-1 activity can be produced within a target cell usinggenetic transformation methods to introduce a modulator encoding nucleicacid molecules into a target cell; and Bap-1 can be inactivated in anon-human mammal to produce animal models of Bap-1 deficiency. Thelatter application, Bap-1 -deficient animals, is particularly useful foridentifying agents that modulate Bap-1 activity and other genes thatencode proteins that interact with Bap-1. The use of nucleic acids forantisense and triple helix therapies and interventions are expresslycontemplated.

The present invention further provides methods of reducing the severityof pathological processes that require integrin mediated signaling.Since association of Bap-1 or Bap-1 complex with a β3 integrin isrequired for integrin-mediated signaling, agents that blockintegrin/Bap-1 association can be used in therapeutic methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B SEQ ID NOs: 1 and 3, respectively, show the nucleic acidsequence of human Bap-1 and the amino acid sequence of human Bap-1.

FIGS. 2A-2B SEQ ID NOs: 3 and 4, respectively, show a partial nucleicacid of mouse Bap-1 and the amino acid sequence of mouse Bap-1.

FIG. 3 shows deletion mutants produced of flag-tagged Bap-1.

FIG. 4 summarizes the effects of the expression of full-length ordeletion mutant, flag-tagged Bap-1 on the expression of a NF-kB-CATfusion.

FIG. 5 summarizes the effects of the expression of full-length ordeletion mutant, flag-tagged Bap-1 on the expression of an AP-1-CATfusion.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

I. General Description

The present invention is based in part on identifying a protein thatbinds to β3 integrins and is involved in integrin-mediated signaling,hereinafter the Bap-1 protein. Bap-1 is also found to associate with thecytoplasmic tail of αIIb and Src kinase.

The Bap-1 protein can be used as an agent, or serve as a target foragents, that can be used to inhibit integrin mediated signaling, forexample to inhibit biological processes requiring GP IIb-IIIa or αVβ3signal transduction.

The present invention is further based on the development of methods forisolating proteins that bind to Bap-1 or a Bap-1/β3 or Bap-1/Src kinasecomplex. Probes based on the Bap-1 protein are used as capture probes toisolate Bap-1/integrin-associated signaling proteins. Dominant negativeproteins, DNAs encoding these proteins, antibodies to these signalingproteins, peptide fragments of these proteins or mimics of theseproteins may be introduced into cells to affect integrin function.Additionally, these proteins provide a novel target for screening ofsynthetic small molecules and combinatorial or naturally occurringcompound libraries to discover novel therapeutics to regulate integrinfunction.

II. Specific Embodiments

A. Bap-1 Protein

The present invention provides isolated Bap-1 protein, as well asallelic variants of the Bap-1 protein, and conservative amino acidsubstitutions of the Bap-1 protein. As used herein, the Bap-1 protein(or Bap-1) refers to a protein that has the amino acid sequence of humanBap-1 depicted in FIG. 1. The Bap-1 protein includes naturally occurringallelic variants of Bap-1, proteins that have a slightly different aminoacid sequence than that specifically recited above. Allelic variants,though possessing a slightly different amino acid sequence than thoserecited above, will still have the requisite ability to associate with aβ3 integrin as part of the relevant signaling cascade.

As used herein, the Bap-1 family of proteins refers to Bap-1 proteinsthat have isolated from organisms in addition to humans. One such memberof the Bap-1 family of proteins is the mouse Bap-1 protein whose partialamino acid and nucleotide sequence is depicted in FIG. 2. The methodsused to identify and isolate other members of the Bap-1 family ofproteins are described below and in Example 10.

As used herein, the Bap family of proteins refers to proteins that bindto β3 integrins, are structurally related to Bap-1, containingsignificant sequence homology to Bap-1. Members of the Bap family ofproteins are involved in integrin-mediated signaling. For convenience,the Bap-1 protein, members of the Bap-1 family of proteins, and membersof the Bap family of proteins are hereinafter referred to as the Bapproteins of the present invention.

The Bap proteins of the present invention are preferably in isolatedform. As used herein, a protein is said to be isolated when physical,mechanical or chemical methods are employed to remove the Bap proteinfrom cellular constituents that are normally associated with the Bapprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated Bap protein.

The Bap proteins of the present invention further include conservativevariants of the Bap proteins herein described. As used herein, aconservative variant refers to alterations in the amino acid sequencethat do not adversely affect the ability of the Bap protein to bind to aβ3 integrin and mediate signaling. A substitution, insertion or deletionis said to adversely affect the Bap protein when the altered sequenceprevents the Bap protein from associating with a β3 integrin. Forexample, the overall charge, structure or hydrophobic/hydrophilicproperties of Bap can be altered without adversely affecting activity ofBap. Accordingly, the amino acid sequence of Bap can be altered, forexample to render the peptide more hydrophobic or hydrophilic, withoutadversely affecting the ability of the peptide to become associated witha β3 protegrin.

Ordinarily, the allelic variants, the conservative substitutionvariants, the members of the Bap family of proteins and especially themembers of the Bap-1 family of proteins, will have an amino acidsequence having at least 75% amino acid sequence identity with the humanor mouse Bap-1 sequence, more preferably at least 80%, even morepreferably at least 90%, and most preferably at least 95%. Identity orhomology with respect to such z sequences is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the known peptides, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. N-terminal, C-terminal or internal extensions,deletions, or insertions into the peptide sequence shall not beconstrued as affecting homology.

Thus, the Bap proteins of the present invention include molecules havingthe amino acid sequences disclosed in FIG. 1; fragments thereof having aconsecutive sequence of at least about 3, 5, 10 or 15 amino acidresidues of the Bap-1 protein; amino acid sequence variants of suchsequence wherein an amino acid residue has been inserted N- orC-terminal to, or within, the disclosed Bap-1 sequence; amino acidsequence variants of the disclosed Bap-1 sequence, or their fragments asdefined above, that have been substituted by another residue.Contemplated variants further include those containing predeterminedmutations by, e.g., homologous recombination, site-directed or PCRmutagenesis, and the corresponding Bap proteins of other animal species,including but not limited to rabbit, rat, murine, porcine, bovine,ovine, equine and non-human primate species, and the alleles or othernaturally occurring variants of the Bap family of proteins; andderivatives wherein the Bap protein has been covalently modified bysubstitution, chemical, enzymatic, or other appropriate means with amoiety other than a naturally occurring amino acid (for example adetectable moiety such as an enzyme or radioisotope).

As described below, members of the Bap family of proteins can beused: 1) to identify and isolate other integrin signaling partners thatbind Bap-1 or a Bap-1 /β3 complex, 2) in methods to identify agents thatblock the association of an integrin with Bap-1 or a Bap-1/signalingcomplex, 3) as a target to assay for integrin mediated signaling, and 4)as a therapeutic agent to block the association of an integrin withBap-1 or a Bap-1/signaling complex.

B. Bap-1 Encoding Nucleic Acid Molecules

The present invention further provides nucleic acid molecules thatencode Bap-1, and the related Bap proteins herein described, preferablyin isolated form. As used herein, "nucleic acid" is defined as RNA orDNA that encodes a peptide as defined above, or is complementary tonucleic acid sequence encoding such peptides, or hybridizes to suchnucleic acid and remains stably bound to it under appropriate stringencyconditions, or encodes a polypeptide sharing at least 75% sequenceidentity, preferably at least 80%, and more preferably at least 85%,with the peptide sequences. Specifically contemplated are genomic DNA,cDNA, mRNA and antisense molecules, as well as nucleic acids based onalternative backbone or including alternative bases whether derived fromnatural sources or synthesized. Such hybridizing or complementarynucleic acid, however, is defined further as being novel and unobviousover any prior art nucleic acid including that which encodes, hybridizesunder appropriate stringency conditions, or is complementary to nucleicacid encoding a Bap protein according to the present invention.

"Stringent conditions" are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0.015M NaCl, 0.0015M sodiumtitrate, 0.1% SDS at 50° C., or (2) employ during hybridization adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C. Another example is use of 50% formamide, 5× SSC 0.75MNaCl 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA(50 Tg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at42° C. in 0.2× SSC and 0.1% SDS. A skilled artisan can readily determineand vary the stringency conditions appropriately to obtain a clear anddetectable hybridization signal.

As used herein, a nucleic acid molecule is said to be "isolated" whenthe nucleic acid molecule is substantially separated from contaminantnucleic acid encoding other polypeptides from the source of nucleicacid.

The present invention further provides fragments of the Bap encodingnucleic acid molecule. As used herein, a fragment of a Bap encodingnucleic acid molecule refers to a small portion of the entire proteinencoding sequence. The size of the fragment will be determined by theintended use. For example, if the fragment is chosen so as to encode anactive portion of the Bap protein, the fragment will need to be largeenough to encode the functional region(s) of the Bap protein. If thefragment is to be used as a nucleic acid probe or PCR primer, then thefragment length is chosen so as to obtain a relatively small number offalse positives during probing/priming. If the fragment is chosen so asto bind the β3 integrin, the length will be chosen so as to contain theβ3 contact site on the Bap protein.

Fragments of the Bap encoding nucleic acid molecules of the presentinvention (i.e., synthetic oligonucleotides) that are used as probes orspecific primers for the polymerase chain reaction (PCR), or tosynthesize gene sequences encoding Bap-1 proteins can easily besynthesized by chemical techniques, for example, the phosphotriestermethod of Matteucci, et al., (J. Am. Chem. Soc. 103:3185-3191, 1981) orusing automated synthesis methods. In addition, larger DNA segments canreadily be prepared by well known methods, such as synthesis of a groupof oligonucleotides that define various modular segments of the Bapgene, followed by ligation of oligonucleotides to build the completemodified Bap gene.

The Bap encoding nucleic acid molecules of the present invention mayfurther be modified so as to contain a detectable label for diagnosticand probe purposes. A variety of such labels are known in the art andcan readily be employed with the Bap encoding molecules hereindescribed. Suitable labels include, but are not limited to, biotin,radiolabeled nucleotides and the like. A skilled artisan can employ anyof the art known labels to obtain a labeled Bap encoding nucleic acidmolecule.

Modifications to the primary structure itself by deletion, addition, oralteration of the amino acids incorporated into the protein sequenceduring translation can be made without destroying the activity of theprotein. Such substitutions or other alterations result in proteinshaving an amino acid sequence encoded by DNA falling within thecontemplated scope of the present invention.

C. Isolation of Other Bap Encoding Nucleic Acid Molecules

As described above, the identification of the human and mouse Bap-1encoding nucleic acid molecules allows a skilled artisan to isolatenucleic acid molecules that encode other members of the Bap-1 family ofproteins in addition to the human and mouse sequence herein described.Further, the presently disclosed nucleic acid molecules allow a skilledartisan to isolate nucleic acid molecules that encode other members ofthe Bap family of proteins in addition to Bap-1.

Essentially, a skilled artisan can readily use the amino acid sequenceof Bap-1 to generate antibody probes to screen expression librariesprepared from cells involved in integrin signaling. Typically,polyclonal antiserum from mammals such as rabbits immunized with thepurified Bap-1 protein (as described below) or monoclonal antibodies canbe used to probe a mammalian cDNA or genomic expression library, such aslambda gt11 library, to obtain the appropriate coding sequence forBap-1, or other members of the Bap family of proteins. The cloned cDNAsequence can be expressed as a fusion protein, expressed directly usingits own control sequences, or expressed by constructions using controlsequences appropriate to the particular host used for expression of theenzyme. FIG. 1 identifies important antigenic and/or putative operativedomains found in the Bap-1 protein sequence. Such regions are preferredsources of antigenic portions of the Bap-1 protein for the production ofprobe, diagnostic, and therapeutic antibodies.

Alternatively, a portion of the Bap-1 encoding sequence herein describedcan be synthesized and used as a probe to retrieve DNA encoding Bap-1families of proteins from any mammalian organisms that possessintegrin-mediated signaling pathways. Oligomers containing approximately18-20 nucleotides (encoding about a 6-7 amino acid stretch) are preparedand used to screen genomic DNA or cDNA libraries to obtain hybridizationunder stringent conditions or conditions of sufficient stringency toeliminate an undue level of false positives.

Additionally, pairs of oligonucleotide primers can be prepared for usein a polymerase chain reaction (PCR) to selectively clone a Bap-encodingnucleic acid molecule. A PCR denature/anneal/extend cycle for using suchPCR primers is well known in the art and can readily be adapted for usein isolating other Bap encoding nucleic acid molecules. FIG. 1identifies regions of the human Bap-1 gene that are particularly wellsuited for use as a probe or as primers.

D. rDNA molecules Containing a Bap Encoding Nucleic Acid Molecule

The present invention further provides recombinant DNA molecules (rDNAs)that contain a Bap encoding sequence. As used herein, a rDNA molecule isa DNA molecule that has been subjected to molecular manipulation insitu. Methods for generating rDNA molecules are well known in the art,for example, see Sambrook et al., Molecular Cloning (1989). In thepreferred rDNA molecules, a Bap encoding DNA sequence is operably linkedto expression control sequences and/or vector sequences.

The choice of vector and/or expression control sequences to which one ofthe Bap encoding sequences of the present invention is operably linkeddepends directly, as is well known in the art, on the functionalproperties desired, e.g., protein expression, and the host cell to betransformed. A vector contemplated by the present invention is at leastcapable of directing the replication or insertion into the hostchromosome, and preferably also expression, of the Bap structural geneincluded in the rDNA molecule.

Expression control elements that are used for regulating the expressionof an operably linked protein encoding sequence are known in the art andinclude, but are not limited to, inducible promoters, constitutivepromoters, secretion signals, and other regulatory elements. Preferably,the inducible promoter is readily controlled, such as being responsiveto a nutrient in the host cell's medium.

In one embodiment, the vector containing a Bap encoding nucleic acidmolecule will include a prokaryotic replicon, i.e., a DNA sequencehaving the ability to direct autonomous replication and maintenance ofthe recombinant DNA molecule extrachromosomally in a prokaryotic hostcell, such as a bacterial host cell, transformed therewith. Suchreplicons are well known in the art. In addition, vectors that include aprokaryotic replicon may also include a gene whose expression confers adetectable marker such as a drug resistance. Typical bacterial drugresistance genes are those that confer resistance to ampicillin ortetracycline.

Vectors that include a prokaryotic replicon can further include aprokaryotic or viral promoter capable of directing the expression(transcription and translation) of the Bap encoding gene sequences in abacterial host cell, such as E. coli. A promoter is an expressioncontrol element formed by a DNA sequence that permits binding of RNApolymerase and transcription to occur. Promoter sequences compatiblewith bacterial hosts are typically provided in plasmid vectorscontaining convenient restriction sites for insertion of a DNA segmentof the present invention. Typical of such vector plasmids are pUC8,pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond,Calif.), pPL and pKK223 available from Pharmacia, Piscataway, N.J.

Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can also be used to form a rDNAmolecules the contains a Bap encoding sequence. Eukaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia),pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC,#31255), the vector pCDM8 described herein, and the like eukaryoticexpression vectors.

Eukaryotic cell expression vectors used to construct the rDNA moleculesof the present invention may further include a selectable marker that iseffective in an eukaryotic cell, preferably a drug resistance selectionmarker. A preferred drug resistance marker is the gene whose expressionresults in neomycin resistance, i.e., the neomycin phosphotransferase(neo) gene. (Southern et al., J. Mol. Anal. Genet. 1:327-341, 1982.)Alternatively, the selectable marker can be present on a separateplasmid, and the two vectors are introduced by co-transfection of thehost cell, and selected by culturing in the appropriate drug for theselectable marker.

E. Host Cells Containing an Exogenously Supplied Bap Encoding NucleicAcid Molecule

The present invention further provides host cells transformed with anucleic acid molecule that encodes a Bap protein of the presentinvention. The host cell can be either prokaryotic or eukaryotic.Eukaryotic cells useful for expression of a Bap-1 protein are notlimited, so long as the cell line is compatible with cell culturemethods and compatible with the propagation of the expression vector andexpression of the Bap-1 gene product. Preferred eukaryotic host cellsinclude, but are not limited to, yeast, insect and mammalian cells,preferably vertebrate cells such as those from a mouse, rat, monkey orhuman fibroblastic cell line. Preferred eukaryotic host cells includeChinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIHSwiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658,baby hamster kidney cells (BHK), and the like eukaryotic tissue culturecell lines.

Any prokaryotic host can be used to express a Bap-encoding rDNAmolecule. The preferred prokaryotic host is E. coli.

Transformation of appropriate cell hosts with a rDNA molecule of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used and host system employed. With regardto transformation of prokaryotic host cells, electroporation and salttreatment methods are typically employed, see, for example, Cohen etal., Proc. Natl. Acad. Sci. USA 69:2110, 1972; and Maniatis et al.,Molecular Cloning, A Laboratory Mammal, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982). With regard to transformation ofvertebrate cells with vectors containing rDNAs, electroporation,cationic lipid or salt treatment methods are typically employed, see,for example, Graham et al., Virol. 52:456, 1973; Wigler et al., Proc.Natl. Acad. Sci. USA 76:1373-76, 1979.

Successfully transformed cells, i.e., cells that contain a rDNA moleculeof the present invention, can be identified by well known techniques.For example, cells resulting from the introduction of an rDNA of thepresent invention can be cloned to produce single colonies. Cells fromthose colonies can be harvested, lysed and their DNA content examinedfor the presence of the rDNA using a method such as that described bySouthern, J. Mol. Biol. 98:503, 1975, or Berent et al., Biotech. 3:208,1985 or the proteins produced from the cell assayed via an immunologicalmethod.

F. Production of Bap using a rDNA molecule encoding a Bap Protein

The present invention further provides methods for producing a Bapprotein that uses one of the Bap encoding nucleic acid molecules hereindescribed. In general terms, the production of a recombinant form of aBap protein typically involves the following steps:

First, a nucleic acid molecule is obtained that encodes a Bap protein,such as the nucleic acid molecule depicted in FIG. 1. If the Bapencoding sequence is uninterrupted by introns, it is directly suitablefor expression in any host.

The Bap encoding nucleic acid molecule is then preferably placed inoperable linkage with suitable control sequences, as described above, toform an expression unit containing the Bap encoding sequences. Theexpression unit is used to transform a suitable host and the transformedhost is cultured under conditions that allow the production of the Bapprotein. Optionally the Bap-1 protein is isolated from the medium orfrom the cells; recovery and purification of the protein may not benecessary in some instances where some impurities may be tolerated.

Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in appropriate hosts. The construction ofexpression vectors that are operable in a variety of hosts isaccomplished using appropriate replicons and control sequences, as setforth above. The control sequences, expression vectors, andtransformation methods are dependent on the type of host cell used toexpress the gene and were discussed in detail earlier. Suitablerestriction sites can, if not normally available, be added to the endsof the coding sequence so as to provide an excisable gene to insert intothese vectors. A skilled artisan can readily adapt any host/expressionsystem known in the art for use with Bap encoding sequences to produceBap protein.

G. Methods to Identify Other Integrin Signaling Partners

Another embodiment of the present invention provides methods for use inisolating and identifying cytoplasmic signaling partners of integrins.Specifically, the Bap protein alone, or in combination with an integrincontaining a β3 subunit (hereinafter a Bap/β3 complex), can be used toidentify signaling partners that bind Bap or Bap/β3 complex from cellsthat express integrins.

In detail, a Bap protein alone, or in combination with an integrincontaining a β3 subunit or Bap/β3 complex, is mixed with an extract orfraction of a cell that expresses an integrin under conditions thatallow the association of a signaling partner with the Bap or Bap/β3complex. After mixing, peptides that have become associated with Bap-1or the Bap-1 /β3 complex are separated from the mixture. The signalingpartner that bound Bap-1 or the Bap-1 /β3 complex can then be removedand further analyzed.

To identify and isolate a signaling partner, the entire Bap protein canbe used. Alternatively, a fragment of a Bap protein can be used.

As used herein, a cellular extract refers to a preparation or fractionwhich is made from a lysed or disrupted cell. The preferred source ofcellular extracts will be cells which naturally express β3 integrins.Examples of such cells include, but are not limited to platelets andleukocytes.

A variety of methods can be used to obtain an extract of a cell. Cellscan be disrupted using either physical or chemical disruption methods.Examples of physical disruption methods include, but are not limited to,sonication and mechanical shearing. Examples of chemical lysis methodsinclude, but are not limited to, detergent lysis and the enzyme lysis. Askilled artisan can readily adapt methods for preparing cellularextracts in order to obtain extracts for use in the present methods.

The cellular extract can be prepared from cells that have been freshlyisolated from a subject or from cells or cell lines which have beencultured. In addition, the extract can be prepared from cells that areeither in a resting state or from cells that have been activated. Avariety of agents can be used to activate a cell. The selection of anactivating agent will be based on the cell type used. For example,thrombin, collagen or ADP can be used to activate platelets while PMAcan be used to activate leukocytes.

Once an extract of a cell is prepared, the extract is mixed with the Bapprotein, or a Bap/β3 complex, under conditions in which association ofthe Bap or Bap/β3 complex with the signaling partner can occur. Avariety of conditions can be used, the most preferred being conditionsthat closely resemble conditions found in the cytoplasm of anintegrin-expressing cell. Features such as osmolarity, pH, temperature,and the concentration of cellular extract used, can be varied tooptimize the association of the integrin with the signaling partner.

After mixing under appropriate conditions, Bap or the Bap/β3 complex isseparated from the mixture. A variety of techniques can be utilized toseparate the mixture. For example, antibodies specific to Bap or theBap/β3 complex can be used to immunoprecipitate the Bap or Bap/β3complex and associated signaling partner. Alternatively, standardchemical separation techniques such as chromatography anddensity/sediment centrifugation can be used.

After removal of nonassociated cellular constituents found in theextract, the signaling partner can be dissociated from the Bap or Bap/β3complex using conventional methods. For example, dissociation can beaccomplished by altering the salt concentration or pH of the mixture.

To aid in separating associated integrin/signaling partner pairs fromthe mixed extract, the Bap or Bap/β3 complex can be immobilized on asolid support. For example, Bap can be attached to a nitrocellulosematrix or acrylic beads. Attachment of Bap to a solid support aids inseparating peptide/signaling partner pair from other constituents foundin the extract.

The identified signaling partners can be either a single protein or acomplex made up of two or more proteins.

Alternatively, the Bap-encoding nucleic acid molecule can be used in ayeast two-hybrid system. The yeast two-hybrid system has been used toidentify other protein partner pairs and can readily be adapted toemploy the Bap encoding molecules herein described. (See Example 2.)

H. Use of Bap and Other Isolated Signaling Partners

Once isolated, the integrin signaling partners obtained using the abovedescribed method, as well as the Bap proteins herein described,especially Bap-1, can be used for a variety of purposes. These proteinscan be used to generate antibodies that bind to the Bap protein, or thesignaling partner, using techniques known in the art. Antibodies thatbind Bap or another integrin signaling partner can be used to assayintegrin signaling, as a therapeutic agent to modulate a biological orpathological process mediated by integrin signaling, or to purify thesignaling partner. These uses are described in detail below.

I. Methods to Identify Agents that Block Integrin Cytoplasmic SignalingPartner Interactions

Another embodiment of the present invention provides methods foridentifying agents that reduce or block the association of an integrinwith a cytoplasmic signaling complex, such as a Bap protein or aBap/signaling partner complex, hereinafter collectively referred to asBap signaling complex. Specifically, a β3 integrin is mixed with a Bapprotein, a cellular extract containing Bap, or a complex of Bap and thesignaling partner described above, in the presence and absence of anagent to be tested. After mixing under conditions that allow associationof the integrin or peptide with the Bap signaling complex, the twomixtures are analyzed and compared to determine if the agent reduced orblocked the association of the integrin with the Bap signaling complex.Agents that block or reduce the association of an integrin with the Bapsignaling complex will be identified as decreasing the amount ofassociation present in the sample containing the tested agent.

In an alternative format, agents that block or reduce thetranscriptional repressor activity of Bap-1 can be isolated by usingfusions between the transcriptional control elements of genes whoseexpression levels are repressed by Bap-1 and reporter genes such as CAT(chloramphenicol acetyl transferase). Examples of such fusions includeNF-kB-CAT, and AP-1-CAT fusions. In this format, cells which express theappropriate reporter fusion are transfected to express Bap-1 in thepresence and absence of the agent to be tested. Cell lines which exhibita reduced ability of Bap-1 to repress the expression of the reportergene in the presence of the agent being tested, identify agents whichare capable of inhibiting Bap-1 repressor activity. Assays to detectreporter gene expression are widely and commercially available as arenumerous reporter genes, including but not limited to CAT andβ-galactosidase (β-gal). For instance, such assays are commerciallyavailable from GIBCO-BRL.

As used herein, an agent is said to reduce or block integrin/Bapsignaling complex association when the presence of the agent decreasesthe extent to which or prevents the Bap signaling complex from becomingassociated with the β3 integrin. One class of agents will reduce orblock the association by binding to the Bap signaling complex whileanother class of agents will reduce or block the association by bindingto the β3 integrin.

The Bap signaling complex used in the above assay can either be anisolated and fully characterized protein, such as Bap-1, or can be apartially characterized protein that binds to Bap-1 or a Bap-1/signalingpartner complex that has been identified as being present in a cellularextract. It will be apparent to one of ordinary skill in the art that solong as the Bap signaling complex has been characterized by anidentifiable property, e.g., molecular weight, the present assay can beused.

Agents that are assayed in the above method can be randomly selected orrationally selected or designed. As used herein, an agent is said to berandomly selected when the agent is chosen randomly without consideringthe specific sequences involved in the association of the integrin withthe Bap signaling complex. An example of randomly selected agents is theuse a chemical library or a peptide combinatorial library, or a growthbroth of an organism.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a nonrandom basis which takes into accountthe sequence of the target site and/or its conformation in connectionwith the agent's action. As described above, there are two sites ofaction for agents that block integrin/Bap signaling complex interaction:the cytoplasmic domain of the β3 subunit or the Bap signaling complex.Agents can be rationally selected or rationally designed by utilizingthe peptide sequences that make up the contact sites of the integrin/Bapsignaling complex pair. For example, a rationally selected peptide agentcan be a peptide whose amino acid sequence is identical to the Bap-1contact site on the cytoplasmic domain of the integrin or the β3 contactsite on Bap-1. Such an agent will reduce or block the association of theintegrin with the signaling partner by binding to Bap-1 or the β3integrin respectively.

The agents of the present invention can be, as examples, peptides, smallmolecules, vitamin derivatives, as well as carbohydrates. A skilledartisan can readily recognize that there is no limit as to thestructural nature of the agents of the present invention.

One class of agents of the present invention are peptide agents whoseamino acid sequences are chosen based on the amino acid sequence of thecytoplasmic domain of the β3 subunit or the amino acid sequence of theBap protein, such as the human Bap-1 sequence depicted in FIG. 1.

The peptide agents of the invention can be prepared using standard solidphase (or solution phase) peptide synthesis methods, as is known in theart. In addition, the DNA encoding these peptides may be synthesizedusing commercially available oligonucleotide synthesis instrumentationand produced recombinantly using standard recombinant productionsystems. The production using solid phase peptide synthesis isnecessitated if non-gene-encoded amino acids are to be included.

Another class of agents of the present invention are antibodiesimmunoreactive with critical positions of the cytoplasmic domain of anintegrin or with a Bap signaling complex such as Bap-1. Antibody agentsare obtained by immunization of suitable mammalian subjects withpeptides, containing as antigenic regions, those portions of the β3cytoplasmic domain or Bap signaling complex, intended to be targeted bythe antibodies. Critical regions include the contact sites involved inthe association of the integrin with the Bap signaling complex.

Antibody agents are prepared by immunizing suitable mammalian hosts inappropriate immunization protocols using the peptide haptens alone, ifthey are of sufficient length, or, if desired, or if required to enhanceimmunogenicity, conjugated to suitable carriers. Methods for preparingimmunogenic conjugates with carriers such as BSA, KLH, or other carrierproteins are well known in the art. In some circumstances, directconjugation using, for example, carbodiimide reagents may be effective;in other instances linking reagents such as those supplied by PierceChemical Co., Rockford, Ill., may be desirable to provide accessibilityto the hapten. The hapten peptides can be extended at either the aminoor carboxy terminus with a Cys residue or interspersed with cysteineresidues, for example, to facilitate linking to a carrier.Administration of the immunogens is conducted generally by injectionover a suitable time period and with use of suitable adjuvants, as isgenerally understood in the art. During the immunization schedule,titers of antibodies are taken to determine adequacy of antibodyformation.

While the polyclonal antisera produced in this way may be satisfactoryfor some applications, for pharmaceutical compositions, use ofmonoclonal preparations is preferred. Immortalized cell lines whichsecrete the desired monoclonal antibodies may be prepared using thestandard method of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the peptide hapten or isthe integrin or signaling complex itself. When the appropriateimmortalized cell culture secreting the desired antibody is identified,the cells can be cultured either in vitro or by production in ascitesfluid.

The desired monoclonal antibodies are then recovered from the culturesupernatant or from the ascites supernatant. Fragments of themonoclonals or the polyclonal antisera which contain the immunologicallysignificant portion can be used as antagonists, as well as the intactantibodies. Use of immunologically reactive fragments, such as the Fab,Fab', of F(ab')₂ fragments is often preferable, especially in atherapeutic context, as these fragments are generally less immunogenicthan the whole immunoglobulin.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of receptor can also be produced in the context ofchimeras with multiple species origin.

The antibodies thus produced are useful not only as modulators of theassociation of an integrin with a Bap signaling complex, but are alsouseful in immunoassays for detecting integrin mediated signaling and forthe purification of integrin-associated signaling proteins.

J. Uses for Agents that Block the Association of an Integrin with a BapSignaling Complex

As provided in the Background section, integrins play important roles inintracellular signaling, cellular attachment, cellular aggregation andcellular migration. Agents that reduce or block the interactions of anintegrin with a Bap signaling complex can be used to modulate biologicaland pathologic processes associated with integrin function and activity.

In detail, a biological or pathological process mediated by an integrincan be modulated by administering to a subject an agent that blocks theinteraction of an integrin with a Bap signaling complex.

As used herein, a subject can be any mammal, so long as the mammal is inneed of modulation of a pathological or biological process mediated byan integrin. The term "mammal" is meant an individual belonging to theclass Mammalia. The invention is particularly useful in the treatment ofhuman subjects.

As used herein, a biological or pathological process mediated by anintegrin or integrin signaling refers to the wide variety of cellularevents in which an integrin binds a substrate producing an intracellularsignal that involves the Bap protein or a Bap signaling complex.Examples of biological processes include, but are not limited to,cellular attachment or adhesion to substrates and other cells, cellularaggregation, cellular migration, cell proliferation, and celldifferentiation.

Pathological processes refer to a category of biological processes whichproduce a deleterious effect. For example, thrombosis is the deleteriousattachment and aggregation of platelets while metastasis is thedeleterious migration and proliferation of tumor cells. Thesepathological processes can be modulated using agents which reduce orblock integrin/Bap signaling complex association.

As used herein, an agent is said to modulate a pathological process whenthe agent reduces the degree or severity of the process. For example, anagent is said to modulate thrombosis when the agent reduces theattachment or aggregation of platelets.

Two known parings of the β3 subunit have been observed: with αV to makeαVβ3, the Vitronectin Receptor; and with GP IIb to make GP IIb-IIIa, theFibrinogen Receptor. αVβ3 is widely distributed, is the most promiscuousmember of the integrin family and mediates cellular attachment to a widespectrum of adhesive proteins, mostly at the R-G-D sequence on theadhesive protein. The biological processes mediated by αVβ3 are diverseand include bone resorption, angiogenesis, tumor metastasis andrestenosis. αVβ3 is known to signal upon adhesive protein ligation (P.I. Leavesley, et al., J. Cell Biol. 121:163-170, 1993). As an example,endothelial cells undergo apoptosis when relieved of ligation (P. C.Brooks, Cell 79:1157-1164, 1994).

GP IIb-IIIa, by contrast, is restricted to platelets and cells ofmegakaryocyte lineage although a report has appeared indicating that GPIIb-IIIa is present in tumor cell lineages. As discussed in detailelsewhere in this application, the function of GP IIb-IIIa is primarilyto bind adhesive proteins to mediate platelet aggregation. In thisfunction, GP IIb-IIIa participates in both inside-out and outside-insignaling. Decreased receptor function of GP IIb-IIIa leads to bleeding;elevated receptor function of GP IIb-IIIa can lead to thrombusformation. Studies have appeared indicating that platelet aggregationthrough GP IIb-IIIa may also be involved in tumor metastasis.

K. Administration of Agents that Affect Integrin Signaling

The agents of the present invention can be provided alone, or incombination with another agents that modulate a particular pathologicalprocess. For example, an agent of the present invention that reducesthrombosis by blocking integrin mediated cellular signaling can beadministered in combination with other anti-thrombotic agents. As usedherein, two agents are said to be administered in combination when thetwo agents are administered simultaneously or are administeredindependently in a fashion such that the agents will act at the sametime.

The agents of the present invention can be administered via parenteral,subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal,or buccal routes. Alternatively, or concurrently, administration may beby the oral route. The dosage administered will be dependent upon theage, health, and weight of the recipient, kind of concurrent treatment,if any, frequency of treatment, and the nature of the effect desired.

The present invention further provides compositions containing one ormore agents which block integrin/signaling complex association. Whileindividual needs vary, determination of optimal ranges of effectiveamounts of each component is within the skill of the art. Typicaldosages comprise 0.1 to 100 Tg/kg body wt. The preferred dosagescomprise 0.1 to 10 Tg/kg body wt. The most preferred dosages comprise0.1 to 1 Tg/kg body wt.

In addition to the pharmacologically active agent, the compositions ofthe present invention may contain suitable pharmaceutically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically for delivery to the site of action. Suitableformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form, for example, water-solublesalts. In addition, suspensions of the active compounds as appropriateoily injection suspensions may be administered. Suitable lipophilicsolvents or vehicles include fatty oils, for example, sesame oil, orsynthetic fatty acid esters, for example, ethyl oleate or triglycerides.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension include, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers. Liposomes can also be used to encapsulate the agentfor delivery into the cell.

The pharmaceutical formulation for systemic administration according tothe invention may be formulated for enteral, parenteral or topicaladministration. Indeed, all three types of formulations may be usedsimultaneously to achieve systemic administration of the activeingredient.

Suitable formulations for oral administration include hard or softgelatin capsules, pills, tablets, including coated tablets, elixirs,suspensions, syrups or inhalations and controlled release forms thereof.

In practicing the methods of this invention, the compounds of thisinvention may be used alone or in combination, or in combination withother therapeutic or diagnostic agents. In certain preferredembodiments, the compounds of this invention may be coadministered alongwith other compounds typically prescribed for these conditions accordingto generally accepted medical practice, such as anticoagulant agents,thrombolytic agents, or other antithrombotics, including plateletaggregation inhibitors, tissue plasminogen activators, urokinase,prourokinase, streptokinase, heparin, aspirin, or warfarin. Thecompounds of this invention can be utilized in vivo, ordinarily inmammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, ratsand mice, or in vitro.

L. Methods for Identifying Integrin-Mediated Signaling

The present invention further provides methods for identifying cellsinvolved in integrin-mediated signaling as well as techniques that canbe applied to diagnose biological and pathological processes associatedwith integrin-mediated signaling. Specifically, integrin-mediatedsignaling can be identified by determining whether the Bap protein, orBap signaling complex, is expressed and/or is associated with a β3integrin. Cells expressing Bap or the Bap signaling complex, or in whichBap or the Bap signaling complex is associated with a β3 integrin areconsidered to be involved in integrin-mediated signaling. Such methodsare useful in identifying sites of inflammation, thrombosis,angiogenesis and tumor metastasis.

In one example, an extract of cells is prepared which contains the β3integrin. The extract is then assayed to determine whether the β3integrin is associated with Bap or a Bap signaling complex. The degreeof association present provides a measurement of the degree of signalingthe cell is participating in. An increase in the degree of signaling isa measurement of the level of integrin mediated activity.

For example, to determine whether a tumor has metastatic potential, anextract is made of the tumor cells and the β3 integrins expressed by thetumor cells are isolated using known methods such asimmunoprecipitation. The integrins are then analyzed, for example, bygel electrophoresis to determine whether a Bap protein is associatedwith the integrin. The presence and level of a Bap associationcorrelates with the metastatic potential of the cancer.

Alternatively, the level of Bap protein or Bap gene expression can beused to directly correlate with the involvement of the cell inintegrin-mediated signaling. A variety of immunological and nucleic acidtechniques can be used to determine if the Bap protein, or a Bapencoding mRNA, is produced in a particular cell. The presence ofincreased levels of the Bap protein or the Bap encoding mRNA, correlateswith the metastatic potential of the cancer.

M. Gene Therapy

The Bap gene and the Bap protein can also serve as a target for genetherapy in a variety of contexts. For example, in one application,Bap-deficient non-human animals can be generated using standardknock-out procedures to inactivate a Bap gene. In such a use, anon-human mammal, for example a mouse or a rat, is generated in which amember of the Bap family of genes is inactivated. This can beaccomplished using a variety of art-known procedures such as targetedrecombination. Once generated, the Bap-deficient animal can be usedto 1) identify biological and pathological processes mediated by Bap, 2)identify proteins and other genes that interact with Bap, 3) identifyagents that can be exogenously supplied to overcome Bap deficiency and4) serve as an appropriate screen for identifying mutations within Bapthat increase or decrease activity.

In addition to animal models, human Bap-deficiency can be corrected bysupplying to a human, a genetic construct that encodes the Bap proteinwhich is deficient in the subject. A variety of techniques are presentlyavailable, and others are being developed, for introducing a nucleicacid molecule into a human subject to correct a genetic deficiency. Suchmethods can be readily adapted to employ the Bap-encoding nucleic acidmolecules of the present invention.

In another embodiment, genetic therapy can be used as a means formodulating a Bap-mediated biological or pathological processes. Forexample, during graft rejection, it may be desirable to introduce intothe subject being treated a genetic expression unit that encodes amodulator of Bap-1/integrin mediated signaling, such as an antisenseencoding nucleic acid molecule. Such a modulator can either beconstitutively produced or inducible within a cell or specific targetcell. This allows a continual or inducible supply of a therapeutic agentwithin the subject.

As set forth in the Examples, Bap-1 appears to be a transcriptionalrepressor that targets genes including those regulated by NF-kB andAP-1. Since both AP-1 and NF-kB are involved in many types of diseasesuch as cancers, the Bap-1 gene itself may serve as a therapeutic agentfor the treatment of diseases related to gene up-regulation.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out preferred embodiments of thepresent invention, and are not to be construed as limiting in any waythe remainder of the disclosure.

Other generic configurations will be apparent to one skilled in the art.

EXAMPLES Example 1 Cloning of Bap-1

A yeast two-hybrid system (Chien et al., Proc. Natl. Acad. Sci. USA88:9578-9582, 1991) was used to identify proteins that interactspecifically with the cytoplasmic tail of the β3 integrin subunit. Thehistidine selection system (Durfee et al., Genes. Dev. 7:555-569, 1993),which utilizes two distinct GAL4-dependent reporter genes, HIS3 and lacZwas employed. The bait was the β3 cytoplasmic domain fused to the GAL4DNA binding domain of vector pGBT9. The new construct was designated aspGBT9-IIIa. Approximately 5×10⁵ primary transformants of mouse embryocDNA library expressed as fusion products with the VP16 activationdomain (Vojtek et al., Cell. 74:205-214, 1993) were screened. Severalhundreds clones showed significant histidine prototrophy and of these 20were strongly β-galactosidase positive. For two of these clones(126-2and 141-1) histidine prototrophy and β-galactosidase activity dependedon the presence of both plasmids. As a further control, segregantscontaining the cDNA library plasmids only were mated with cellsexpressing GAL4 DNA binding protein only (pGBT9) and several tester GAL4DNA binding protein fusions including v-Raf and mutant v-Raf (Li et al.,EMBO 14:685-696, 1995). Histidine prototrophy and β-galactosidaseactivity were recovered only in combination with original pGBT9-IIIa.

The cDNA library plasmid DNA of 126-2 and 141-1 were transformed into E.coli and subjected to DNA sequencing. The sequence data showed that theinserts of these two clones are from one single gene, and a search ofthe GenBank database revealed that this gene is not represented in thedatabase. DNA sequencing showed that neither of the cDNA clones containa complete protein coding region. To clone the full-length cDNA of the126-2 human homologue, a human bone marrow 5O-stretch plus 1 gt11 cDNAlibrary (Clontech, CAT HL5005b) were screened using a 400 bp NotIfragment isolated from clone 126-2 as probe. Approximately 5×10⁵ cloneswere screened and two positive clones, gt5 and gt6, were identified. Thetwo clones were amplified using standard techniques. DNA were preparedand the inserts were subcloned into pBSKSII, cut at the NotI site. Theclone containing the NotI fragment from gt5 was designated pBSgt5, andthe clone containing the NotI insert from phage gt6 was designatedpBSgt6. Restriction mapping and preliminary sequencing indicated thatpBSgt6 has an insert of 3.5 kbp, and pBSgt5 has an insert of 2.1 kbp,contained within pBSgt6. The cDNA insert of the pBSgt6 was subjected tosequencing analysis, and was shown to have a size of 3.5 kbp containingan open reading frame.

Example 2 Bap-1 Interacts with αIIb and v-Src Kinase in the YeastTwo-Hybrid System

A spectrum of proteins of the integrin family or mutants, and somepotential signaling molecules, were tested for their ability to interactwith Bap-1 in the yeast two-hybrid system. Bap-1 was demonstrated tointeract with the cytoplasmic tail of αIIb and v-Src tyrosine kinase,but not with the cytoplasmic tail of β2, β3(S752P) serine to prolinemutant, or a β3/β1 chimera. Further, Bap-1 was seen to interact withBap-1 in the two-hybrid, suggesting that a functional homodimer may beformed within cells. The results indicate that Bap-1 might link integrinαIIbβ3 signal transduction by bridging other known signaling moleculessuch as pp60src kinase. Src kinase, a major platelet tyrosine kinase, isspeculated to be involved in tyrosine-specific phosphorylation ofcellular proteins during platelet activation by different agonists. Srckinase was shown to phosphorylate the β3 integrin in vitro (Law et al.,J. Biol. Chem. 271:10811-10815, 1996). 40% of total platelet pp60srcbecomes associated with the cytoskeletal fraction upon plateletactivation (Horvath et al., EMBO J. 11:855-861, 1992). Thrombinstimulation of platelets induces a transient increase in the specificactivity of pp60c-src followed by a redistribution of pp60csrc to thetriton X-100-insoluble, cytoskeleton-rich fraction (Clark et al., Molec.Cell Biol. 13:1863-1871, 1993). This association requires plateletaggregation and actin polymerization (Oda et al., J. Biol. Chem.267:20075-20081, 1992). The present findings implicate Bap-1 as involvedin both outside-in and inside-out signaling by bridging integrins withprotein tyrosine kinases.

Example 3 Analysis of Mouse Bap-1

Bap-1 encodes a protein of 336 amino acids with a RING finger domain.Analysis of the protein sequence of the Bap-1 identifies an unusualamino-terminal RING finger motif that can be written as CX2CX11ECLHXFCX2CX11CX2(SEQ ID NO:5). The proteins that share a RING domainhave been reported to be implicated during development, such as DG17(Driscoll and Williams, MCB 7:4482-4489, 1987) and Posterior Sex Combsand Suppressor two of zeste (Brunk et al., Nature 353:351-353, 1991;Lohuizen et al., Nature 353:353-355, 1991), gene transcription such asRPT-1 (Patarca et al., Proc. Natl. Acad. Sci. 85:2733-2737, 1988), DNArepair such as RAD-18 (Jones et al., Nucleic Acids Res. 16: 7119-7131,1988), oncogenic transformation such as BMI-1 (Haupt et al., Cell65:753-763, 1991), tumor suppression such as BRCA-1 (Miki et al.,Science 266:66-71, 1994), and signal transduction such as CD40-bindingprotein (CD40-bp) (Hu et al., J. Biol. Chem. 269:30069-30072, 1994) andTRAF2 (Rothe et al., Cell 78:681-692, 1994; Hsu et al., Cell 84:299-308,1996). The solution structure of the RING finger domain from the acutepromyelocyte leukemia pro-oncoprotein PML suggested that the PML RINGfinger is involved in making protein-protein interactions (Borden etal., 1995. EMBO J. 14: 1532-1541), a molecular mechanism which iscommonly used in signaling protein complexes.

Bap-1 has significant sequence similarity with the ring 1 gene, ie., ithas 48% identity with RING1 gene at amino acid level, the function ofwhich is not known. Interestingly, Bap-1 shares 17% identity withDrosophila gene Posterior Sex Combs (Psc) that is believed to behomologous to oncogene bmi-1, that was originally found to be involvedin B- and T- cell lymphoma. Psc is a member of the Polycomb-group genefamily, which is required to maintain the repression of homeotic genesthat regulate the identities of Drosophila segments. bmi-1 appeared toplay a similar role in vertebrates: bmi-1 knock-out mice shows posteriortransformations of the axial skeleton (van der Lugt et al., Genes & Dev.8: 757-769, 1994); and overexpression of bmi-1 in mice shows theopposite phenotype, a dose-dependent anterior transformation ofvertebral identity (Aldema et al., Nature 374:724-727, 1995). Both thePsc and bmi-1 can repress activator function when transiently introducedinto cells (Bunker and Kingston, Molec. Cell Biol. 14:1721-1732, 1994).Furthermore mel-18, another Polycomb group-related mammalian gene whichshares an amino acid sequence including RING-finger motif, can functionas a transcriptional negative regulator with tumor suppressor activity.

All this suggests that the Bap-1 protein may play an important role incell migration, cell proliferation, and development, for which the roleof integrins is implicated. For example, the Bap-1 protein may functionas a latent transcriptional regulator, either positive or negative, thatis inactive when bound to the β3 cytoplasmic tail of integrins, but canbe activated by outside-in or inside-out signaling and then dissociatesfrom the tail and translocates into the nucleus.

Example 4 Bap-1 Exhibits Transcriptional Repressor Activity

To determine the effects of Bap-1 expression on the transcriptionalcontrol of Ap-1 and NF-kB dependent promoters, flag-tagged, full lengthbap-1 as well as deletion mutants of bap-1 were transfected into NIH3T3cells containing CAT fusions to Ap-1 and NF-kB dependent transcriptionalcontrol elements. Bruder et al. (1992) Genes & Dev., 6, 545-556, andBaldwin et al. (1991) Mol. Cell Biol., 11, 4943-4951. Deletion mutantsof bap-1 including the encoded amino acids are set forth in FIG. 3.

FIG. 4 summarizes the effects of the expression of full length anddeletion mutants of bap-1 on the expression of the NF-kB-CAT reporterfusion in NIH3T3 cells. NIH3T3 cells were grown in 100 mm dishes to 70%confluence in DMEM with 10% calf serum and transfected with plasmid DNA(10 μg/dish each of pCIneoBap-1 or its deletion mutant, together with 4μg/dish of reporter plasmids, pMHC-NF-κB-CAT or pB4X) usingLipofectAMINE (GIBCO-BRL), following manufacturer's instruction. 24hours after transfection, cells were washed twice with PBS; DMEM with0.25% calf serum was added and incubated for additional 12 hours. Cellswere then trypsinized and resuspended in DMEM containing 0.25% calfserum and overlayed on 0.7% agarose. After cells were incubated as suchfor 6 hours at 37° C. in a CO₂ incubator, half of the cells weretransferred to dishes coated with vitronectin at 5 μg/ml (for Adhesion)and half of the cells remained in the 0.7% agar (for suspension). After90 minutes, cells were harvested and CAT ELISA assays were performedaccording to vendor's instruction (Boehringer Mannheim, cat#1363727).Expression of full-length Bap-1 results in a slight decrease in theexpression of the NF-kB-CAT fusion. Deletion of the RING domain fromBap-1 results in an apparent inability of Bap-1 to exhibittranscriptional repression of the NF-kB-CAT fusion. Deletion of theC-terminal end of Bap-1 results in the strong transcriptionalsuppression of the NF-kB-CAT fusion. These results suggest that Bap-1RING domain is involved in repression activity, and that the C-terminalhalf of the protein is involved in negatively regulating the repressoractivity.

FIG. 5 summarizes the effects of the expression of full length anddeletion mutants of bap-1 on the expression of the AP-1-CAT reporterfusion in NIH3T3 cells. Cell culture and transfections were done asabove. Expression of full-length Bap-1 results in a slight decrease inthe expression of the AP-1-CAT fusion. Deletion of the RING domain fromBap-1 results in an apparent decreased ability of Bap-1 to exhibittranscriptional repression of the AP-1-CAT fusion. Deletion of theC-terminal end of Bap-1 results in the strong transcriptionalsuppression of the AP-1-CAT fusion. These results suggest thatfull-length Bap-1 is involved in inhibiting AP-1 reporter activity.Since the wild-type Bap-1 appears to be ineffective in regulating NF-κB,this suggests that wild-type Bap-1 may specifically regulating certaingenes. Since the RING deletion demonstrates repressor activity, althoughimpaired, this suggests that in the protein there is a second regionthat is involved in repressor activity. Consistent with the dataobtained with the NF-kB reporter, the C-terminal half of the protein isinvolved in regulating the repressor activity. Deletion of theC-terminal half of the protein will render the protein constitutivelyactive.

Example 5 Tissue distribution of Bap-1

The tissue distribution of Bap-1 expression was examined by Northernblot, and it was found that Bap-1 is expressed in most tissues,consistent with a suggestion that Bap-1 has an important role incellular functions. In addition to the 3.6 kb mRNA, we detected a 2.4 kbsize mRNA in most tissues, which might be a spliced form of Bap-1, or aBap- related gene.

Clone 126-2, the partial cDNA of the mouse homologue of Bap-1, waslocated in the central part of the Bap-1, and is 98% identical withBap-1. Again, the high degree of conservation suggest a fundamental rolefor Bap-1 in cellular regulation.

Interaction between Bap-1 and the cytoplasmic tail observed in the yeasttwo-hybrid is unlikely to be mediated by a yeast protein. This wasfurther demonstrated by an in vitro binding assay using purified αIIbβ3and purified Bap-1 protein. Specifically, Bap-1 was expressed as aGST-Bap-1 fusion protein in E. coli, and purified and immobilized onglutathione agarose beads. The immobilized GST-Bap-1 fusion was thenincubated with purified αIIbβ3, and retention on the beads was analyzedby SDS-PAGE and Western Blotting. The results showed that β3 proteinbinds specifically to GST-Bap-1, but not to GST controls. The ability toconfirm the binding between Bap-1 and the β3 cytoplasmic tail in vitrois consistent with the robust interaction observed in the yeasttwo-hybrid system.

Example 6 Expression of Bap-1 in a Heterologous System

CHO cells expressing αIIbβ3, like in platelets, manifest highlyregulated changes in the ligand binding affinity as measured by PAC-1binding. The CHO cell heterologous system facilitated the analysis ofrecombinant gene functions in the role of regulating the αIIbβ3affinity. An example of this type of analysis is that CHO cellsexpressing αIIbβ3 were shown to be activated by recombinant R-Ras.(Zhang et al., Cell 85:61-69, 1996) To look at the effects of Bap-1 onthe regulation of αIIbβ3, αIIbβ3 is co-expressed with the Bap-1proteins, and deletion variants of Bap-1, and the cellular phenotypesare examined accordingly.

Two different strategies can be used to reduce endogenous Bap-1activity/production; namely using an antisense Bap-1 molecule or usingan inhibitory mutant of Bap-1. The RING domain in the TRAF2 and PMLproteins have been implicated as involving protein-protein interactions.In the case of TRAF2, the RING deletion mutant function as adominant-negative inhibitor of TNF-mediated NF-kB activation (Cell84:299-308). Accordingly, RING deletion may block or activate Bap-1depending on the nature of the molecules that associate with the Bap-1RING domains. To this end, Bap-1 and variants thereof, have beenflag-tagged and inserted into mammalian expression vectors. Stable celllines that express the wild-type Bap-1, Bap-1 variants (such as the RINGdeletion mutant Bap-1), the Bap-1 anti-sense RNA, and the vector controlcan be generated using standard transformation methods. The expressionof Bap-1 can be determined by anti-flag and anti-Bap-1 antibodies. Thecell lines can then be examined for the effect on the activation ofαIIbβ3 (PAC-1 binding), cell spreading, and attachment on fibrinogen.Over-expression of a signaling molecule such as Bap-1 can desensitizethe regulatory pathway. Therefore, transient and/or conditionalexpression can also be exploited to characterize the functions of theBap-1 gene.

Example 7 Effects of Bap-1 on the αVβ3 Functions on Melanoma

Since the expression of Bap-1 is not restricted to αIIbβ3 expressingcells such as platelets, Bap-1 is likely to be involved in the αVβ3functions. To this end, Bap-1 and variants thereof can be transfectedinto an αVβ3 expressing cell line such as M21 melanoma. Again, theadhesive properties such as attachment and spreading on vitronectin canbe examined. The expression of αVβ3 in M21 melanoma is essential formetastasis.

Example 8 The Cellular Localization of Bap-1

The cellular localization of Bap can be examined by in situimmunohistochemistry. Anti-Bap-1 monoclonal antibodies, particularlyantibodies generated against 1) a GST-Bap-1 fusion protein expressed andpurified in E. coli, 2) a 26 mer N-terminal peptide corresponding to aunique region of Bap-1 protein, and/or 3) a 26 mer peptide correspondingto the central part of the protein can be readily generated and used inthe immunohistochemical examination of Bap-1 production/expression. Thetissue distribution of Bap-1 can be examined using these Bap-1antibodies.

Example 9 Bap-1/β3 Interaction In Vivo

To confirm the interaction observed in the yeast two-hybrid systemdescribed above and in vitro, anti-Bap-1 antibodies can be used toimmunoprecipitate Bap-1 and SDS-PAGE can be used for furtherpurification. Then the presence of β3 can be detected by Westernblotting using anti-β3 antibodies. The reverse experiment can also beperformed by immunoprecipitating β3 and blotting with anti-flag oranti-Bap-1 antibodies.

Example 10 Identification of the Domains and Amino Acids that areCritical for Protein-Protein Interaction

The critical residues both in the β3 tail and in the Bap-1 protein canbe further identified by deletion and mutagenesis analysis. In oneapplication, these experiments are done in a heterologous cell using theyeast two-hybrid system described above.

Example 11 Cloning of Bap-1 Related Genes by PCR or Library Screening

A messenger RNA of 2.4 kb in size, in addition to the 3.5 kb Bap-1 mRNA,was observed in most tissues examined by Northern blot using the humanBap-1 cDNA or the mouse partial cDNA as probe. These results suggestedthat there are Bap-1 related genes or spliced forms expressed. The Bap-1related genes can be readily isolated using Bap-1 cDNA as probe toscreen cDNA libraries, genomic libraries, or can be used to designprimers based on the Bap-1 DNA sequence. The isolated genes can then beexamined for their functions in integrin signaling as detailed in theother examples listed in this application.

Genomic studies of Bap-1 gene: The human chromosomal Bap-1 gene can beisolated by conventional approaches, and its genomic structure andchromosome location can be determined. A genomic database can besearched to determine if its chromosome location corresponds to anydisease locus.

Example 12 Oncogenic or Tumor Suppressor Activity of Bap-1

Oncogenic transformation is often accompanied by deregulation ofintegrin signaling, such as an increase or decrease of cell adhesion. Itis interesting to note that Bap-1 has amino acid similarity toproto-oncogene Bmi-1 and tumor suppressor gene Mel-18. Accordingly, theoncogenic or tumor suppressor activity of Bap-1 can be tested. Briefly,Bap-1 and its variants are transfected into Rat-1 or NIH3T3 cells, andsoft agar growth and focus formation are scored, both of which areindicative of oncogenic activity. To test tumor suppressor activity ofBap-1, Bap-1 and its variants are co-transfected with a series of knownoncogenes into Rat-1 or NIH3T3 cells, and the effects of Bap-1 on theoncogenic transformation of known oncogenes are examined.

Tumor metastasis requires angiogenesis, and integrin αVβ3 is essentialfor angiogenesis. By blocking αVβ3 signaling such as by modulatingBap-1/β3 interaction, tumor metastasis can be inhibited.

Example 13 Generation of Bap-1 Homozygous Deficient Mice

Routine genetic procedures can be used to create genetic knock-outmutants of mice in which Bap-1 has been inactivated. The preferredmethod is to introduce, using targeted homologous recombination, anucleic acid molecule that contains multiple stop codons in each readingframe. This serves to inactivate the Bap-1 locus. Such mice can be usedto further study biochemical and physiological effects of Bap-1.

Although the present invention has been described in detail withreference to examples above, it is understood that various modificationscan be made without departing from the spirit of the invention.Accordingly, the invention is limited only by the following claims. Allcited patents and publications referred to in this application areherein incorporated by reference in their entirety.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 5                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3467 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 226..1233                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GCCCCTCGCT CGCTCGCTCC TTCCCGCCCT CCCCGCAGCG CCGGCCGAGC CG -             #GCTTCCCC     60                                                                 - - TCAGTCTCTC ATGAATATTG AGCGGCCCCT GTTGTATTTC CCGAGCTCCA TT -            #GCGGAAGC    120                                                                 - - TGAGGCTCGC CATATTGTGC GGCGGCGCCG GCGTCCGCGG CAGCTGATAC CA -            #GAGTCTTG    180                                                                 - - CTCCGGCCGC GGCCAGCGGA GCCCTGGGCT GGGGCAGGAG CCGCA ATG - #TCT CAG            234                                                                                         - #                  - #              Met Ser G - #ln                         - #                  - #                1                    - - GCT GTG CAG ACA AAC GGA ACT CAA CCA TTA AG - #C AAA ACA TGG GAA CTC          282                                                                       Ala Val Gln Thr Asn Gly Thr Gln Pro Leu Se - #r Lys Thr Trp Glu Leu                 5            - #      10            - #      15                          - - AGT TTA TAT GAG TTA CAA CGA ACA CCT CAG GA - #G GCA ATA ACA GAT GGC          330                                                                       Ser Leu Tyr Glu Leu Gln Arg Thr Pro Gln Gl - #u Ala Ile Thr Asp Gly            20                 - # 25                 - # 30                 - # 35       - - TTA GAA ATT GTG GTT TCA CCT CGA AGT CTA CA - #C AGT GAA TTA ATG TGC          378                                                                       Leu Glu Ile Val Val Ser Pro Arg Ser Leu Hi - #s Ser Glu Leu Met Cys                            40 - #                 45 - #                 50              - - CCA ATT TGT TTG GAT ATG TTG AAG AAC ACC AT - #G ACT ACA AAG GAG TGT          426                                                                       Pro Ile Cys Leu Asp Met Leu Lys Asn Thr Me - #t Thr Thr Lys Glu Cys                        55     - #             60     - #             65                  - - TTA CAT CGT TTT TGT GCA GAC TGC ATC ATC AC - #A GCC CTT AGA AGT GGC          474                                                                       Leu His Arg Phe Cys Ala Asp Cys Ile Ile Th - #r Ala Leu Arg Ser Gly                    70         - #         75         - #         80                      - - AAC AAA GAA TGT CCT ACC TGT CGG AAA AAA CT - #A GTT TCC AAA AGA TCA          522                                                                       Asn Lys Glu Cys Pro Thr Cys Arg Lys Lys Le - #u Val Ser Lys Arg Ser                85             - #     90             - #     95                          - - CTA AGG CCA GAC CCA AAC TTT GAT GCA CTC AT - #C AGC AAA ATT TAT CCA          570                                                                       Leu Arg Pro Asp Pro Asn Phe Asp Ala Leu Il - #e Ser Lys Ile Tyr Pro           100                 1 - #05                 1 - #10                 1 -      #15                                                                              - - AGT CGT GAT GAG TAT GAA GCT CAT CAA GAG AG - #A GTA TTA GCC AGG        ATC      618                                                                    Ser Arg Asp Glu Tyr Glu Ala His Gln Glu Ar - #g Val Leu Ala Arg Ile                          120  - #               125  - #               130              - - AAC AAG CAC AAT AAT CAG CAA GCA CTC AGT CA - #C AGC ATT GAG GAA GGA          666                                                                       Asn Lys His Asn Asn Gln Gln Ala Leu Ser Hi - #s Ser Ile Glu Glu Gly                       135      - #           140      - #           145                  - - CTG AAG ATA CAG GCC ATG AAC AGA CTG CAG CG - #A GGC AAG AAA CAA CAG          714                                                                       Leu Lys Ile Gln Ala Met Asn Arg Leu Gln Ar - #g Gly Lys Lys Gln Gln                   150          - #       155          - #       160                      - - ATT GAA AAT GGT AGT GGA GCA GAA GAT AAT GG - #T GAC AGT TCA CAC TGC          762                                                                       Ile Glu Asn Gly Ser Gly Ala Glu Asp Asn Gl - #y Asp Ser Ser His Cys               165              - #   170              - #   175                          - - AGT AAT GCA TCC ACA CAT AGC AAT CAG GAA GC - #A GGC CCT AGT AAC AAA          810                                                                       Ser Asn Ala Ser Thr His Ser Asn Gln Glu Al - #a Gly Pro Ser Asn Lys           180                 1 - #85                 1 - #90                 1 -      #95                                                                              - - CGG ACC AAA ACA TCT GAT GAT TCT GGG CTA GA - #G CTT GAT AAT AAC        AAT      858                                                                    Arg Thr Lys Thr Ser Asp Asp Ser Gly Leu Gl - #u Leu Asp Asn Asn Asn                          200  - #               205  - #               210              - - GCA GCA ATG GCA ATT GAT CCA GTA ATG GAT GG - #T GCT AGT GAA ATT GAA          906                                                                       Ala Ala Met Ala Ile Asp Pro Val Met Asp Gl - #y Ala Ser Glu Ile Glu                       215      - #           220      - #           225                  - - TTA GTA TTC AGG CCT CAT CCC ACA CTT ATG GA - #A AAA GAT GAC AGT GCA          954                                                                       Leu Val Phe Arg Pro His Pro Thr Leu Met Gl - #u Lys Asp Asp Ser Ala                   230          - #       235          - #       240                      - - CAG ACG AGA TAC ATA AAG ACT TCT GGT AAC GC - #C ACT GTT GAT CAC TTA         1002                                                                       Gln Thr Arg Tyr Ile Lys Thr Ser Gly Asn Al - #a Thr Val Asp His Leu               245              - #   250              - #   255                          - - TCC AAG TAT CTG GCT GTG AGG TTA GCT TTA GA - #A GAA CTT CGA AGC AAA         1050                                                                       Ser Lys Tyr Leu Ala Val Arg Leu Ala Leu Gl - #u Glu Leu Arg Ser Lys           260                 2 - #65                 2 - #70                 2 -      #75                                                                              - - GGT GAA TCA AAC CAG ATG AAC CTT GAT ACA GC - #C AGT GAG AAG CAG        TAT     1098                                                                    Gly Glu Ser Asn Gln Met Asn Leu Asp Thr Al - #a Ser Glu Lys Gln Tyr                          280  - #               285  - #               290              - - ACC ATT TAT ATA GCA ACA GCC AGT GGC CAG TT - #C ACT GTA TTA AAT GGC         1146                                                                       Thr Ile Tyr Ile Ala Thr Ala Ser Gly Gln Ph - #e Thr Val Leu Asn Gly                       295      - #           300      - #           305                  - - TCT TTT TCT TTG GAA TTG GTC AGT GAG AAA TA - #C TGG AAA GTG AAC AAA         1194                                                                       Ser Phe Ser Leu Glu Leu Val Ser Glu Lys Ty - #r Trp Lys Val Asn Lys                   310          - #       315          - #       320                      - - CCC ATG GAA CTT TAT TAC GCA CCT ACA AAG GA - #G CAC AAA TGAGCCTTTA          1243                                                                       Pro Met Glu Leu Tyr Tyr Ala Pro Thr Lys Gl - #u His Lys                           325              - #   330              - #   335                          - - AAAACCAATT CTGAGACTGA ACTTTTTTAT AGCCTATTTC TTTAATATTA AA -             #GATGTACT   1303                                                                 - - GGCATTACTT TTATGGAGAT CTTGGATATG TTGTTCAATT TTCTTTCTGA GC -            #CAGACTAG   1363                                                                 - - TTTACGCTAT TCAAATCTTT TCCCCTTTAT TTAAGATTTC CTTTTTGGAA GG -            #GACTGCAA   1423                                                                 - - TTATTCAGTA TTTTTTTCTT TCCTTTAAAA AAATATATCT GAAGTTTCTT GT -            #GTTTTTTT   1483                                                                 - - TTTTCCCCAC AAAGTGTGTT TCCACTTGGA GCACCATTTT GACCCAGGAA TT -            #TTTCATAG   1543                                                                 - - TTTCTGTATT CTTATAAGAT TCAGTTGGCT GTCCTTTTCC TGCTCCCCTC AA -            #AAGATTTT   1603                                                                 - - TAGTCATACA GAATGTTAAA TATTATGTAT TCTGACTTTT TTTTTCCCCC GG -            #AGTCTTGT   1663                                                                 - - ATATTTATAG TTTTCCTATA TAAACTGTAG TATCTTCATG AAGAACCCAA GG -            #CTCAAATT   1723                                                                 - - TACTGTCCTT AAAAACAATT CTCATAGGAT TATTCTTTTC ATGGTATTTT CT -            #TCCATAAT   1783                                                                 - - ATCTCATTTT AAAAAGAAGT TCTTTATGAA ACTTAGTGTC CATTGTCATG CA -            #ATGTTTTT   1843                                                                 - - TTTTTCCATT CTTTTTCCCC TGTAATTTTG GAATTTCTGG TCCTGGGAAG AA -            #TCAAACAA   1903                                                                 - - AATCTTAAGT TCTATGAGAA CTTGGTTCAT TGACATATTC TGCTGAAGAA AG -            #AAAAATTA   1963                                                                 - - AATTGGTAGT AAAATATAGT CTTCAAGTAT ACGTTTGAGA GTGCTTTTTT TT -            #GTATTAGT   2023                                                                 - - TCTGCTGTCA CTTCATTTCC TGTATTATAT GTGATGTTTT TCCCCATTAA AA -            #TACCAGAG   2083                                                                 - - ATAATGGAGA TATTTTGCAC TTTAGCCTTG ATGAAAAGTA CAAGATATGT TC -            #AAAGCTTC   2143                                                                 - - CCTAATTTTT TTCTTATTTG TAGCCACATA AGTTTCAAGA ATAACATGGC AC -            #ACAGAACA   2203                                                                 - - ATGGAAAAAA GTTTGTTTCC ATTGGAAAAT TATATCATTT TGGGTTGCCA CA -            #TCAGTTTA   2263                                                                 - - TAAATTTGGC GCTCTTTTAA TTACACTCTG TAGAAGGTTA ATAGAGCTTG AG -            #CCCTGCTT   2323                                                                 - - TAATATGTAG TGAAAGATAA TTCTGTAGAA AAACGTCAGC CAGTAGGGTA AA -            #GTCATTCT   2383                                                                 - - ACTGTTCTTA ATTTTTATAT TGAGGAACAA TATTGGGTGT TTGGGAGCCA GA -            #AAGCTTTG   2443                                                                 - - TTGACAGATC AGAAATAAGA TTGACTTGGG TGTTATATTT CATCTCTCTC CA -            #GACTCTAG   2503                                                                 - - GTATATTTCC AACTTTATAT ATCACAGTAT TTAAAAAGAC ATGTTTGCAT TG -            #AGAAATTA   2563                                                                 - - ACCCTAAAGG GTTTTCAATA GGGTGTAGAC CTCCAGTACC TTTGTAACTA AA -            #GTCTGTCT   2623                                                                 - - AGTCATTGTA AATATTTATC TGTCAGTTTT GACAGATTGG GGCCAGCTTG AT -            #GTTTTAAA   2683                                                                 - - TCTTCAGCCC GGTATGAAAA CTTAAAGGTA TATATTCAAT TTTTTACCAT TT -            #TATGGAAA   2743                                                                 - - ATATTTAAAA TTTGTTTTTA CAGGGTTTTT TTTTTTTTTT TTTTTTTTTT GT -            #AATCTGTG   2803                                                                 - - CCATGAAATT TGAAAACCAC CAAAAATCAA GGGAACTTTT ATATATTCAA TT -            #CCTTTTCT   2863                                                                 - - GGTGTAATGT TAAAGTTGTA TAGATTATTA ATGCATGCCC ACTGAATATA AC -            #CCTGGTTT   2923                                                                 - - TGTGATAAAA CTGCTTAGAT TTTGTTGATG ACATTAGATT AGTAGTTGCA TT -            #AAATAACT   2983                                                                 - - AAATTCCCAT TGTGATTAAT TGAAATTTTG TCTTTAAGCA GAGAGTTATT TG -            #TGACTATA   3043                                                                 - - AGCTTTGTGC TTAGAGAATG TATGTGTTTT TATCTGTCAG TATGGGAGGA TA -            #TAAACTGC   3103                                                                 - - ATCATTAGTG AAATTATTGG TTGTGTAATC CTTTGTGAAA TATAATTCTA GG -            #TATTTGAT   3163                                                                 - - AGGGTATTGA GTGTATTTTG TGTGTGTGTG GATGTGTGTT TTGGGGTACG GG -            #GAGAGGCG   3223                                                                 - - ATGCTATTGG CCATCACTAC CAACCAGGGT TTCAAAAAGT ATATACCTAA GT -            #AATTTCTT   3283                                                                 - - TTATCACTAC CTCAACTGAG GAAGAAAAGG CTCACCACAA GTGGTGTGAA GG -            #CTTTGGGT   3343                                                                 - - ACTTAGTTCT AAATTTTTTT ATGGTAACAT ATACATAGCC ACATTTACAG TT -            #TTAACCAT   3403                                                                 - - TTTAAGGCAT GTAATTCAGT GGGGTTAGGT ACATTCACAA TGTTGTGTAA TG -            #ATCACCGC   3463                                                                 - - CGTG                 - #                  - #                  - #               3467                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 336 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Ser Gln Ala Val Gln Thr Asn Gly Thr Gl - #n Pro Leu Ser Lys Thr        1               5 - #                 10 - #                 15              - - Trp Glu Leu Ser Leu Tyr Glu Leu Gln Arg Th - #r Pro Gln Glu Ala Ile                   20     - #             25     - #             30                  - - Thr Asp Gly Leu Glu Ile Val Val Ser Pro Ar - #g Ser Leu His Ser Glu               35         - #         40         - #         45                      - - Leu Met Cys Pro Ile Cys Leu Asp Met Leu Ly - #s Asn Thr Met Thr Thr           50             - #     55             - #     60                          - - Lys Glu Cys Leu His Arg Phe Cys Ala Asp Cy - #s Ile Ile Thr Ala Leu       65                 - # 70                 - # 75                 - # 80       - - Arg Ser Gly Asn Lys Glu Cys Pro Thr Cys Ar - #g Lys Lys Leu Val Ser                       85 - #                 90 - #                 95              - - Lys Arg Ser Leu Arg Pro Asp Pro Asn Phe As - #p Ala Leu Ile Ser Lys                  100      - #           105      - #           110                  - - Ile Tyr Pro Ser Arg Asp Glu Tyr Glu Ala Hi - #s Gln Glu Arg Val Leu              115          - #       120          - #       125                      - - Ala Arg Ile Asn Lys His Asn Asn Gln Gln Al - #a Leu Ser His Ser Ile          130              - #   135              - #   140                          - - Glu Glu Gly Leu Lys Ile Gln Ala Met Asn Ar - #g Leu Gln Arg Gly Lys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Gln Gln Ile Glu Asn Gly Ser Gly Ala Gl - #u Asp Asn Gly Asp        Ser                                                                                             165  - #               170  - #               175             - - Ser His Cys Ser Asn Ala Ser Thr His Ser As - #n Gln Glu Ala Gly Pro                  180      - #           185      - #           190                  - - Ser Asn Lys Arg Thr Lys Thr Ser Asp Asp Se - #r Gly Leu Glu Leu Asp              195          - #       200          - #       205                      - - Asn Asn Asn Ala Ala Met Ala Ile Asp Pro Va - #l Met Asp Gly Ala Ser          210              - #   215              - #   220                          - - Glu Ile Glu Leu Val Phe Arg Pro His Pro Th - #r Leu Met Glu Lys Asp      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asp Ser Ala Gln Thr Arg Tyr Ile Lys Thr Se - #r Gly Asn Ala Thr        Val                                                                                             245  - #               250  - #               255             - - Asp His Leu Ser Lys Tyr Leu Ala Val Arg Le - #u Ala Leu Glu Glu Leu                  260      - #           265      - #           270                  - - Arg Ser Lys Gly Glu Ser Asn Gln Met Asn Le - #u Asp Thr Ala Ser Glu              275          - #       280          - #       285                      - - Lys Gln Tyr Thr Ile Tyr Ile Ala Thr Ala Se - #r Gly Gln Phe Thr Val          290              - #   295              - #   300                          - - Leu Asn Gly Ser Phe Ser Leu Glu Leu Val Se - #r Glu Lys Tyr Trp Lys      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Val Asn Lys Pro Met Glu Leu Tyr Tyr Ala Pr - #o Thr Lys Glu His        Lys                                                                                             325  - #               330  - #               335             - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 328 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..327                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - AGT GGA GCA GAA GAT AAT GGT GAC AGC TCC CA - #C TGT AGT AAC GCA TCC           48                                                                       Ser Gly Ala Glu Asp Asn Gly Asp Ser Ser Hi - #s Cys Ser Asn Ala Ser                       340      - #           345      - #           350                  - - ACA CAC AGC AAC CAG GAA GCG GGC CCG AGT AA - #C AAA CGG ACC AAA ACC           96                                                                       Thr His Ser Asn Gln Glu Ala Gly Pro Ser As - #n Lys Arg Thr Lys Thr                   355          - #       360          - #       365                      - - TCT GAT GAC TCT GGG CTT GAT CTT GAT AAC AA - #C AAT GCA GGA GTG GCG          144                                                                       Ser Asp Asp Ser Gly Leu Asp Leu Asp Asn As - #n Asn Ala Gly Val Ala               370              - #   375              - #   380                          - - ATT GAT CCA GTC ATG GAC GGT GCC AGT GAG AT - #T GAG TTA GTC TTC AGG          192                                                                       Ile Asp Pro Val Met Asp Gly Ala Ser Glu Il - #e Glu Leu Val Phe Arg           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - CCC CAT CCA ACT CTT ATG GAA AAG GAC GAC AG - #C GCA CAG ACG AGA        TAC      240                                                                    Pro His Pro Thr Leu Met Glu Lys Asp Asp Se - #r Ala Gln Thr Arg Tyr                          405  - #               410  - #               415              - - ATA AAG ACT TCA GGC AAT GCC ACT GTT GAT CA - #C TTA TCC AAG TAT CTG          288                                                                       Ile Lys Thr Ser Gly Asn Ala Thr Val Asp Hi - #s Leu Ser Lys Tyr Leu                       420      - #           425      - #           430                  - - GCT GTG AGG TTA GCT TTA GAA GAA CTT CGA AG - #C AAA GTG A                 - #   328                                                                    Ala Val Arg Leu Ala Leu Glu Glu Leu Arg Se - #r Lys Val                               435          - #       440          - #       445                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 109 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Ser Gly Ala Glu Asp Asn Gly Asp Ser Ser Hi - #s Cys Ser Asn Ala Ser        1               5 - #                 10 - #                 15              - - Thr His Ser Asn Gln Glu Ala Gly Pro Ser As - #n Lys Arg Thr Lys Thr                   20     - #             25     - #             30                  - - Ser Asp Asp Ser Gly Leu Asp Leu Asp Asn As - #n Asn Ala Gly Val Ala               35         - #         40         - #         45                      - - Ile Asp Pro Val Met Asp Gly Ala Ser Glu Il - #e Glu Leu Val Phe Arg           50             - #     55             - #     60                          - - Pro His Pro Thr Leu Met Glu Lys Asp Asp Se - #r Ala Gln Thr Arg Tyr       65                 - # 70                 - # 75                 - # 80       - - Ile Lys Thr Ser Gly Asn Ala Thr Val Asp Hi - #s Leu Ser Lys Tyr Leu                       85 - #                 90 - #                 95              - - Ala Val Arg Leu Ala Leu Glu Glu Leu Arg Se - #r Lys Val                              100      - #           105                                         - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Glu      1               5   - #                10  - #                15               - - Cys Leu His Xaa Phe Cys Xaa Xaa Cys Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa                  20      - #            25      - #            30                   - - Xaa Xaa Xaa Xaa Cys Xaa Xaa                                                      35                                                                   __________________________________________________________________________

What is claimed:
 1. An isolated protein comprising the amino acidsequence of either SEQ ID NO:2 or SEQ ID NO.:4.
 2. An isolated proteinconsisting of the amino acid sequence selected from the group consistingof: SEQ ID NO:2 and SEQ ID NO:4.
 3. An isolated protein comprising aprotein which is encoded by a nucleic acid whose complement hybridizesto SEQ ID NO:1 under conditions of sufficient stringency after washingat 42° C. in 0.2× SSC and 0.1% SDS to produce a clear signal, whereinsaid isolated protein interact with integrin β3 cytoplasmic tails.
 4. Anisolated protein comprising a protein which is encoded by a nucleic acidwhose complement hybridizes to SEQ ID NO:3 under conditions ofsufficient stringency after washing at 42° C. in 0.2× SSC and 0.1% SDSto produce a clear signal, wherein said isolated protein interacts withintegrin β3 cytoplasmic tails.
 5. An isolated protein comprising aprotein which contains at least one conservative amino acid substitutionof SEQ ID NO: 2, wherein said protein interacts with integrin β3cytoplasmic tails.
 6. An isolated protein comprising a protein whichcontains at least one conservative amino acid substitution of SEQ ID NO:4, wherein said protein interacts with integrin β3 cytoplasmic tails.